Cardiac Compositions

ABSTRACT

Embodiments of the present invention provide a composition comprising perivascular stem cells or induced pluripotent cells (iPS) and a NELL-1 factor for cardiac or vascular regeneration and methods of making and using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/US2010/002298, filed on Aug. 20, 2010, which claims the benefit of U.S. provisional application No. 61/235,618, filed on Aug. 10, 2009. The teaching in these applications is incorporated hereto in its entirety by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention generally relates to cardiac compositions comprising perivascular stem cells or induced pluripotent stem cells and NELL-1 protein and methods of making and using the same.

BACKGROUND OF THE INVENTION

Cardiovascular disease accounts for 1 in every 2.8 deaths in the United States and costs approximately $475 billion in 2009 [1]. 56% of these deaths come from diseases of peripheral and coronary arteries, the latter resulting in 500,000 coronary artery bypass grafts (CABG) each year [1]. Together with CABG, lower limb revascularization and arteriovenous access grafts for hemodialysis account for more than 1 million vascular graft implantations per year [2]. Although autologous grafts are the gold standard for bypass procedures, they are limited by availability. Current synthetic grafts are not suitable for small-diameter (ID<6mm) vascular applications due to the acute thrombosis [3-5]. While a tissue-engineered vascular graft (TEVG), constructed by incorporating cells within a biodegradable scaffold, seems to be a possible solution to this challenge [6-8], its success greatly relies on an appropriate cell source and an efficient cellular delivery and carrier system.

Mesenchymal stem cells (MSCs) exhibiting multipotentiality and self-renewal capabilities could overcome the limitations of slow growth rate and phenotype switching with terminally-differentiated vascular cells during in vitro culture [9,10]. However, MSCs have shortcomings, e.g., heterogenicity and limited availability.

The embodiments described below address the above identified problems and needs.

SUMMARY OF THE INVENTION

In an aspect of the present invention, it is provided a composition. The composition comprises a population of perivascular stem cells (PSC) or induced pluripotent stem cells (iPS) and a cardioinductive agent. The cardioinductive agent can be a chemical or biological agent in a therapeutically effective amount for causing PSC or iPS to differentiate in the cardiac cell or progenitor lineages so as to generate cardio tissues. Alternatively, the cardioinductive agent can be a chemical or biological agent in a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth so as to treat, delay, or ameliorate a cardiac condition.

In some embodiments, the cardioinductive agent is a NELL-1 factor, which can be a NELL-1 protein. The NELL-1 protein is in a therapeutically effective amount in the composition, which upon delivery, is effective for causing PSC or iPS to differentiate into a cardiac cell or progenitor cell to achieve cardio tissue regeneration so as to treat, delay, or ameliorate a cardiac or cardiovascular condition. Alternatively, the NELL-1 protein is in a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth so as to treat, delay, or ameliorate a cardiac condition.

The PSC can be human PSC or animal PSC, and can be autologous PSC, allograft PSC, or xenograft PSC. The iPS can be human iPS or animal iPS, and can be autologous iPS, allograft iPS, or xenograft iPS. The PSC or iPS can have a density from about 1×10⁴ to about 1×10⁸/mL (per 1 mL volume of the composition). In some embodiments, the seeding density can be from about 1×10⁴ to about 1×10⁶, from about 1×10⁴ to about 1×10⁵, from about 1×10⁵ to about 1×10⁷, from about 1×10⁵ to about 1×10⁶, from about 1×10⁶ to about 1×10⁷/ml, or from about 1×10⁷to about 1×10⁸/ml. Examples of seeding densities can be, e.g., 0.5×10⁴, 1×10⁴, 0.5×10⁵, 1×10⁵, 0.5×10⁶, 1×10⁶, 0.5×10⁷, 1×10⁷, or 1×10⁸/ml.

In some embodiments, the term PSC can be pericytes or adventitia cells.

The composition can be formulated into different formulations. In some embodiments, the composition can be a scaffold. The scaffold can include one or more excipients. In some embodiments, the scaffold can include a plurality of pores, which can load the PSC or iPS. In some embodiments, an agent such as NELL-1 protein can be embedded in the body of scaffold, and the PSC or iPS is seeded in the pores in the scaffold. In some embodiments, the composition can be a vascular graft. In some embodiments, the composition can be biodegradable or biodurable.

In another aspect of the present invention, it is provided an implantable device. The implantable device comprises a composition of the various embodiments above.

In some embodiments, the implantable device can be a stent. In some embodiments, the stent can have a supporting body and an optional coating, wherein the PSC or iPS and NELL-1 protein are included in the supporting body or the optional coating.

In some embodiments, the support body or the optional coating of the stent can comprise a plurality of pores, wherein the NELL-1 protein is embedded in the support body or the optional coating, and wherein the PSC or iPS is seeded in the pores.

In some embodiments, the optional coating of the stent can be biodegradable or biodurable.

In some embodiments, the stent itself can be biodegradable or biodurable.

In another aspect of the present invention, it is provided a method of fabricating a composition, comprising. The method comprises:

providing a population of perivascular stem cells (PSC) or induced pluripotent stem cells (iPS),

providing a cardioinductive agent, and

forming the composition,

wherein the cardioinductive agent is in (1) a therapeutically effective amount for causing PSC or iPS to differentiate in the cardiac cell or progenitor lineages so as to generate cardio tissues or (2) a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth.

The composition is according to the various embodiments described above and below.

Some embodiments of the present are drawn to methods of fabricating a implantable device. Such methods generally comprise:

providing a population of PSC or iPS,

providing a cardioinductive agent, and

forming the implantable device.

wherein the cardioinductive agent is in (1) a therapeutically effective amount for causing PSC or iPS to differentiate in the cardiac cell or progenitor lineages so as to generate cardio tissues or (2) a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth.

The implantable device is according to the various embodiments described above and below.

In another aspect of the present invention, it is provided a method of treating or ameliorating a cardiac condition, comprising administering to a subject a composition according to the various embodiments described above and below or a implantable device according to according to the various embodiments described above and below. Examples of such cardiac conditions include cardiovascular conditions or myocardial infarction (MI).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of experiments showing increased pericyte proliferation/survival when Nell-1 is added.

FIG. 2 shows the results of experiments showing increased VEGF expression by pericytes when Nell-1 is added.

DETAILED DESCRIPTION OF THE INVENTION

In an aspect of the present invention, it is provided a composition. The composition comprises a population of perivascular stem cells (PSC) or induced pluripotent stem cells (iPS) and a cardioinductive agent. The cardioinductive agent can be a chemical or biological agent in a therapeutically effective amount for causing PSC or iPS to differentiate in the cardiac cell or progenitor lineages so as to generate cardio tissues. Alternatively, the cardioinductive agent can be a chemical or biological agent in a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth so as to treat, delay, or ameliorate a cardiac condition.

In some embodiments, the cardioinductive agent is a NELL-1 factor, which can be a NELL-1 protein. The NELL-1 protein is in a therapeutically effective amount in the composition, which upon delivery, is effective for causing PSC or iPS to differentiate into a cardiac cell or progenitor cell to achieve cardio tissue regeneration so as to treat, delay, or ameliorate a cardiac or cardiovascular condition. Alternatively, the NELL-1 protein is in a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth so as to treat, delay, or ameliorate a cardiac condition.

The PSC can be human PSC or animal PSC, and can be autologous PSC, allograft PSC, or xenograft PSC. The iPS can be human iPS or animal iPS, and can be autologous iPS, allograft iPS, or xenograft iPS. The PSC or iPS can have a density from about 1×10⁴ to about 1×10⁸/mL (per 1 mL volume of the composition). In some embodiments, the seeding density can be from about 1×10⁴to about 1×10⁶, from about 1×10⁴to about 1×10⁵, from about 1×10⁵to about 1×10⁷, from about 1×10⁵to about 1×10⁶, from about 1×10⁶to about 1×10⁷/ml, or from about 1×10⁷to about 1×10⁸/ml. Examples of seeding densities can be, e.g., 0.5×10⁴, 1×10⁴, 0.5×10⁵, 1×10⁵, 0.5×10⁶, 1×10⁶, 0.5×10⁷, 1×10⁷, or 1×10⁸/ml.

In some embodiments, the term PSC can be pericytes or adventitia cells.

The composition can be formulated into different formulations. In some embodiments, the composition can be a scaffold. The scaffold can include one or more excipients. In some embodiments, the scaffold can include a plurality of pores, which can load the PSC or iPS. In some embodiments, an agent such as NELL-1 protein can be embedded in the body of scaffold, and the PSC or iPS is seeded in the pores in the scaffold. In some embodiments, the composition can be a vascular graft. In some embodiments, the composition can be biodegradable or biodurable.

In another aspect of the present invention, it is provided a implantable device. The implantable device comprises a composition of the various embodiments above.

In some embodiments, the implantable device can be a stent. In some embodiments, the stent can have a supporting body and an optional coating, wherein the PSC or iPS and NELL-1 protein are included in the supporting body or the optional coating.

In some embodiments, the support body or the optional coating of the stent can comprise a plurality of pores, wherein the NELL-1 protein is embedded in the support body or the optional coating, and wherein the PSC or iPS is seeded in the pores.

In some embodiments, the optional coating of the stent can be biodegradable or biodurable.

In some embodiments, the stent itself can be biodegradable or biodurable.

In another aspect of the present invention, it is provided a method of fabricating a composition, comprising. The method comprises:

providing a population of perivascular stem cells (PSC) or induced pluripotent stem cells (iPS),

providing a cardioinductive agent, and

forming the composition,

wherein the cardioinductive agent is in (1) a therapeutically effective amount for causing PSC or iPS to differentiate in the cardiac cell or progenitor lineages so as to generate cardio tissues or (2) a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth.

The composition is according to the various embodiments described above and below.

Some embodiments of the present are drawn to methods of fabricating a implantable device. Such methods generally comprise:

providing a population of PSC or iPS,

providing a cardioinductive agent, and

forming the implantable device.

wherein the cardioinductive agent is in (1) a therapeutically effective amount for causing PSC or iPS to differentiate in the cardiac cell or progenitor lineages so as to generate cardio tissues or (2) a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth.

The implantable device is according to the various embodiments described above and below.

In another aspect of the present invention, it is provided a method of treating or ameliorating a cardiac condition, comprising administering to a subject a composition according to the various embodiments described above and below or a implantable device according to according to the various embodiments described above and below. Common cardiac conditions can be acute or chronic cardiac conditions, which are described in more detail below. Examples of such cardiac conditions include cardiovascular conditions or myocardial infarction (MI).

As used herein, the term “therapeutically effective amount” means the dose of a cardioinductive agent (e.g., a NELL-1 factor) required to cause a PSC or iPS to differentiate into a cardio tissue cell or progenitor cell to achieve cardio tissue regeneration so as to treat, delay, or ameliorate a cardiac condition. Alternatively, the term “therapeutically effective amount” means the dose of a cardioinductive agent (e.g., a NELL-1 factor) required for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth so as to treat, delay, or ameliorate a cardiac condition, for example, decreasing of the injury and/or accelerating the healing of ischemic or infracted myocardium.

As used herein, the terms “NELL-1 protein”, “NELL-1 peptide” and “NELL-1 factor” are sometimes used interchangeably.

As used herein, the term “cardioinductive agent” refers to any chemical agent or biological agent that is either (1) effective for causing PSC or iPS to differentiate into a cardiac cell or progenitor cell to achieve cardio tissue regeneration so as to treat, delay, or ameliorate a cardiac or cardiovascular condition or (2) effective for helping the PSC survive or engraft better in a cardiac tissue. An example of cardioinductive agent is NELL-1 protein.

Cell-Based Cardiac Regeneration

Cardiac tissue regeneration can be achieved using mesenchymal stem cells (MSC) technology (see, e.g., Gersh, et al., Cardiac Cell Repair Therapy: a Clinical Perspective, in Mayo Clin Proc. 84(10):872-892 (2009) (Review)). Various methods of cardiac tissue regeneration using mesenchymal stem cells such as U.S. patent application publication Nos. 20090123435 and 20070014773. PSC is reportedly a perivascular ancestor of MSCs (see, e.g., Crisan, et al., Cell Stem Cell 3:301-33 (2008); Corselli, et al., Arterioscler Thromb Vasc Biol 30:1104-1109 (2010)). PSC based vascular regeneration is documented. For example, PSC has been seeded in a synthetic vascular graft to achieve vascular regeneration (data not shown).

Advantages of using PSC rather than MSCs for cardiac tissue regeneration are multi-facets. The majority of cell expansion or culturing procedures are based on the use of fetal bovine serum (FBS), which carries the risk of xenoimmunization [e.g., nonhuman immunogenic sialic acid (Neu5Gc)] and transmission of known (e.g., prions transmitting bovine spongiform encephalopathy) and unknown pathogens. Also, irrespective of culture medium, ex vivo culture increases the risk of microbiological or particulate contamination as well as genetic instability (Dahl, J. A. et al. Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum. Int J Dev Biol 52, 1033-42 (2008)). From a safety standpoint, the use of freshly harvested and sorted PSC is superior to cultured MSC such as ASC because it decreases immunogenic, infectious, and tumorigenic risks.

Other advantages of PSC use over MSC are: 1) precise characterization in terms of native tissue localization, phenotype and developmental potential, (MSC are retrospectively derived from primary, heterogeneous cell cultures) and 2) improved trophic potency (we have determined that PSC secrete 10-20 times more heparin binding epidermal growth factor and 3-7 times more basic fibroblast growth factor and vascular endothelial growth factor than classically derived adipose tissue and cord blood MSC (Chen, C. W. et al. Perivascular multilineage progenitor cells in human organs: Regenerative units, cytokine sources, or both? Cytokines and Growth Factors Reviews (2009)). Thus from an FDA regulatory perspective using PSC will facilitate demonstration of product identity, purity, sterility, safety, and potency.

As used herein, the term perivascular stem cells (PSC) shall encompass pericyte and adventitia cells.

Isolation of PSC are well documented. For example, pericyte cells were isolated from various tissues by Peault and Huard in U.S. application Ser. No. 11/746,979. Isolation of adventitial cells from various tissues are well documented (data not shown).

PSC have been isolated from essentially all tissues tested including skeletal muscle, pancreas, placenta, adipose, brain, heart, skin, lung, eye, gut, bone marrow, umbilical cord, or teeth. In some embodiments, autologous PSC can be purified through fluorescence activated cell sorting (FACS) from the stromal vascular fraction of adipose tissues in numbers sufficient to achieve clinical efficacy without ex vivo expansion. The following describes an example of isolating pericyte PSC from human skeletal muscle tissues:

Isolation of pericytes. Briefly, skeletal muscle is separated from fat and macro vasculature then minced into small pieces. The muscle is then incubated (e.g., for 45 min at 37° C.) in medium containing DMEM high glucose (Gibco), 20% FBS (Gibco), 1% Penicillin-Streptomycin (PS) (Gibco) and complemented by 0.5 mg/ml of each collagenases type I, II, and IV. The resulting cell suspension was filtered to eliminate all debris. After rinsing, cells are FACS-sorted according to positive expression for CD 146, NG2 (a proteoglycan associated with pericytes during vascular morphogenesis) and PDGF-R13, and to the absence of hematopoietic (CD45), endothelial (CD34), and myogenic (CD56) cell markers. The dead cells are excluded by FACS via propidium iodure staining. Sorted pericytes are seeded (e.g., at 2×10⁴ cells/cm² in endothelial cell growth medium 2 (EGM-2, Cambrex Bioscience)) and cultured (e.g., at 37° C. for 2 weeks in plates coated with 0.2% gelatin (Calbiochem)). Pericytes are trypsinized once a week and cultured (e.g., at 1:3 dilution (from passage 1 to 5) then at 1:10 (after passage 5)). Except for the first passage, all pericytes are cultured (e.g., in DMEM/FBS/PS proliferation medium) in uncoated flasks to maintain their original phenotype. Pericytes between passages 9 and 11 are used for all tests. Isolation of adventitia PSC is exemplified by the procedure described in Example 1.

NELL-1 Factor

“A NELL-1 factor” as used herein, includes wild type (i.e., naturally occurring) Nell 1 proteins of any mammalian origin, such as human, murine, rat and the like. Exemplary NELL-1 factors for use in the present invention include human NELL-1 protein (SEQ ID NO: 1), murine NELL-1 protein (SEQ ID NO: 2), and rat NELL-1 protein (SEQ ID NO: 3).

“A NELL-1 factor” as used herein, also includes functional derivatives of a wild type NELL-1 protein. A “functional derivative” refers to a modified NELL-1 protein which has one or more amino acid substitutions, deletions or insertions as compared to a wild type NELL-1 protein, and which retains substantially the activity of a wild type NELL-1 protein. By “substantially” is meant at least 50%, at least 75%, or even at least 85% of the activity of a wild type NELL-1 protein. According to the present invention, in order for the functional derivative to substantially retain the activity or function of a wild type NELL-1 protein, the functional NELL-1 derivative shares a sequence identity with the wild type NELL-1 protein of at least 75%, at least 85%, at least 95% or even 99%.

The structure of NELL-1 proteins has been characterized (see, e.g., Kuroda et al., 1999a; Kuroda et al., 1999b, Desai et al., 2006). For example, the murine NELL-1 protein (SEQ ID NO: 4) is a protein of 810 amino acids, having a secretion signal peptide (amino acids #1 to 16), an N-terminal TSP-like module (amino acids #29 to 213), a Laminin G region (amino acids #86 to 210), von Willebrand factor C domains (amino acids #273 to 331 and 699 to 749), and a Ca²⁺-binding EGF-like domains (amino acids #549 to 586).

The secretion signal peptide domain of NELL-1 protein is an amino acid sequence in the protein that is generally involved in transport of the protein to cell organelles where it is processed for secretion outside the cell. The N-terminal TSP-like module is generally associated with heparin binding. von Willebrand factor C domains are generally involved with oligomerization of NELL-1. Laminin G domains of NELL-1 protein are generally involved in adherence of NELL-1 protein to specific cell types or other extracellular matrix proteins. The interaction of such domains with their counterparts is generally associated with, for example, processes such as differentiation, adhesion, cell signaling or mediating specific cell-cell interactions in order to promote cell proliferation and differentiation. The Ca²⁺-binding EGF-like domains of NELL-1 binds protein kinase C beta, which is typically involved in cell signaling pathways in growth and differentiation.

The amino acid sequence of NELL-1 protein is very highly conserved, especially across mammalian species. For example, the murine NELL-1 protein shares about 93% sequence identity with the human NELL-1 protein (SEQ ID NO: 1), which, in turn, shares about 90% sequence identity with the rat NELL-1 protein (SEQ ID NO: 2). Those skilled in the art can use any of the well-known molecular cloning techniques to generate NELL-1 derivatives having one or more amino acid substitutions, deletions or insertions, taking into consideration the functional domains (e.g., secretion signal peptide sequence, N-terminal TSP-like module, Laminin G region, von Willebrand factor C domain) of NELL-1. See, for example, Current Protocols in Molecular Cloning (Ausubel et al., John Wiley & Sons, New York).

The minimum length of a NELL-1 functional derivative is typically at least about 10 amino acids residues in length, more typically at least about 20 amino acid residues in length, even more typically at least about 30 amino acid residues in length, and still more typically at least about 40 amino acid residues in length. As stated above, wild type NELL-1 protein is approximately about 810 amino acid residues in length. A NELL-1 functional derivative can be at most about 810 amino acid residues in length. For example, a NELL-1 functional derivative can be at most at most about 820, 805, 800, 790, 780, 750, 600, 650 600, 550, etc. amino acid residues in length.

Once a NELL-1 protein derivative is made, such protein can be tested to determine whether such derivative retains substantially the activity or function of a wild type NELL-1 protein. For example, the ability of a NELL-1 derivative to bind PKC beta can be tested. Suitable assays for assessing the binding of NELL-1 to PKC beta is described in e.g., Kuroda et al. (1999b). For example, protein-protein interaction can be analyzed by using the yeast two-hybrid system. Briefly, a modified NELL-1 protein can be fused with GAL4 activating domain and the regulatory domain of PKC can be fused with the GAL4 DNA-binding domain. The activity of beta-galactosidase in yeast cells can be detected.

In addition, one can also test the ability of a NELL-1 derivative to stimulate differentiation of precursor cells, which are in the cardiomyocyte lineage, towards mature cardiomyocytes. Maturity of cardiomyocytes can be assessed cellularly (histology) and molecularly (expression of cardiac-specific proteins or extracellular matrix materials). Still further, as an alternative assay, a NELL-1 derivative can be tested for its ability to drive osteoblast precursors to mature bone cells, by detecting expression of late molecular bone markers or mineralization (i.e., calcium deposits). By comparing the activity of a NELL-1 derivative with that of a wild type NELL-1 protein in one or more of the assays such as those described above, one should be able to determine whether such derivative retains substantially the activity or function of a wild type NELL-1 protein.

A NELL-1 protein or functional derivative thereof may be prepared by methods that are well known in the art. One such method includes isolating or synthesizing DNA encoding the NELL-1 protein, and producing the recombinant protein by expressing the DNA, optionally in a recombinant vector, in a suitable host cell, including bacterial, yeast, insect or mammalian cells. Such suitable methods for synthesizing DNA are, for example, described by Caruthers et al. 1985. Science 230:281-285 and DNA Structure, Part A: Synthesis and Physical Analysis of DNA, Lilley, D. M. J. and Dahlberg, J. E. (Eds.), Methods Enzymol., 211, Academic Press, Inc., New York (1992).

NELL-1 protein is effective for enhancing survivability and engraftment of PSC or iPS. Studies on such effects of NELL-1 on pericytes were performed and described in Example 2, the results of which are shown in FIG. 1. FIG. 1 shows increased pericyte proliferation/survival when Nell-1 is added. NELL-1 is also effective for causing PSC or iPS to differentiate into cardiac cell or progenitor cell lineage so as to generate a cardio tissue. Studies on such effects are described in Example 2, the results of which are shown in FIG. 2. FIG. 2 shows increased VEGF expression by pericytes when Nell-1 is added.

The term “therapeutically effective amount” as used here means the dose of a NELL-1 factor required to cause a PSC or iPS to differentiate into a cardio tissue cell or progenitor cell to achieve cardio tissue regeneration so as to treat, delay, or ameliorate a cardiac condition. Alternatively, the term “therapeutically effective amount” means the dose of a NELL-1 factor required for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth so as to treat, delay, or ameliorate a cardiac condition, for example, decreasing of the injury and/or accelerating the healing of ischemic or infracted myocardium.

Precise dosages depend on the cardiac tissue cell type, disease state or condition being treated and other clinical factors, such as weight and condition of the subject, the subject's response to the therapy, the type of formulations and the route of administration. As a general rule, a suitable dose of a NELL-1 composition (i.e., including a NELL-1 protein or nucleic acid) for the administration to adult humans ranges from about 0.001 mg to about 20 mg per kilogram of body weight. In some embodiments, a suitable dose of a NELL-1 composition for the administration to adult humans is in the range of about 0.01 mg to about 5 mg per kilogram of body weight. However, the precise dosage to be therapeutically effective and non-detrimental can be determined by those skilled in the art.

NELL-1 protein and method of making the protein has been described in U.S. application Ser. Nos. 10/544,553, 11/392,294, 11/713,366, and 11/594,510, and U.S. Pat. No. 7,052,856. U.S. patent application publication No. 20090087415 describes a pharmaceutical composition comprising a NELL-1 protein in a therapeutically effective amount for treating a cardiovascular disorder. The teachings in these applications and patent are incorporated herein by reference.

Formulations and Carriers

The composition disclosed herein can be formulated into different formulations. The composition can include materials and carriers to effect a desired formulation. For example, the composition can include an injectable or moldable material that can set within a pre-defined period of placement. Such a pre-defined period can be, e.g., 10 minutes, 30 minutes, one hour, two hours, etc.

In some embodiments, the composition can include a chemical gel that includes primary bonds formed due to changes in pH, ionic environment, and solvent concentration. Examples of such chemical gels can be, but are not limited to, polysaccharides such as chitosan, chitosan plus ionic salts such as beta-glycerophosphates, alginates plus Ba²⁺, Sr²⁺, Ca²⁺, Mg²⁺, collagen, fibrin, plasma or combinations thereof.

In some embodiments, the composition can include a physical gel that include secondary bonds formed due to temperature changes. Examples of such physical gels can be, but are not limited to, alginate, poly(ethylene glycol)-poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) (PEG-PLGA-PEG) tri-block copolymers, agarose, and celluloses. In some embodiments, physical gels that can be used in the composition described herein can include physical gels that are liquid under high shear but gels to solid at low shear. Examples of such physical gels include, but are not limited to, hyaluronic acid, or polyethylene oxides. The physical gels can have pre-formed materials with pre-defined dimensions and shape.

In some embodiments, the composition described herein can include a material that degrade or release active agents in response to a stimulus. Some examples of such stimuli are mechanical stimuli, light, temperature changes, pH changes, change of ionic strength, or electromagnetic field. Such materials are known in the art. Some examples of such materials are chitosan, alginates, pluronics, methyl cellulose, hyaluronic acids, and polyethylene oxides. Other examples are described by Brandl F, Sommer F, Goepferich A. “Rational design of hydrogels for tissue engineering: Impact of physical factors on cell behavior “in Biomaterials. Epub 2006 Sep. 29.

In some embodiments, the composition described herein including any of the gels described above can further include a crosslinker to further tailor degradation kinetics and controlled release. Alternatively, in some embodiments, the composition described herein can include an interpenetrating phase composite or interpenetrating network (IPN) that includes any of the above described gels. Some examples of the crosslinker includes, but are not limited to, common crosslinking agents (polyalkylene oxide, ethylene dimethacrylate, N,N′-methylenebisacrylamide, methylenebis(4-phenyl isocyanate), ethylene dimethacrylate, divinylbenzene,allyl methacrylate, carbodiimidazole, sulfonyl chloride, chlorocarbonates, n-hydroxysuccinimide ester, succinimidyl ester, epoxides, aryl halides, sulfasuccinimidyl esters, and maleimides); PEG based crosslinkers (e.g. MAL-dPEGx-NHS-esters, MAL-dPEGx acid, Bis-MAL-dPEGx, etc.) and photo/light activated crosslinkers, N-hydroxysuccinimide-based crosslinkers, dilysine, trilysine, and tetralysine.

The composition described herein can include a carrier. The carrier can be a polymeric carrier or non-polymeric carrier. In some embodiments, the carrier can be biodegradable, such as degradable by enzymatic or hydrolytic mechanisms. Examples of carriers include, but are not limited to synthetic absorbable polymers such as such as but not limited to poly(α-hydroxy acids) such as poly (L-lactide) (PLLA), poly (D, L-lactide) (PDLLA), polyglycolide (PGA), poly(lactide-co-glycolide (PLGA), poly(-caprolactone), poly(trimethylene carbonate), poly(p-dioxanone), poly(-caprolactone-co-glycolide), poly(glycolide-co-trimethylene carbonate) poly (D, L-lactide-co-trimethylene carbonate), polyarylates, polyhydroxybutyrate (PHB), polyanhydrides, poly(anhydride-co-imide), propylene-co-fumarates, polylactones, polyesters, polycarbonates, polyanionic polymers, polyanhydrides, polyester-amides, poly(amino-acids), homopolypeptides, poly(phosphazenes), poly(glaxanone), polysaccharides, and poly(orthoesters), polyglactin, polyglactic acid, polyaldonic acid, polyacrylic acids, polyalkanoates; copolymers and admixtures thereof, and any derivatives and modifications. See for example, U. S. Pat. No. 4,563,489, and PCT Int. Appl. No. WO/03024316, herein incorporated by reference. Other examples of carriers include cellulosic polymers such as, but not limited to alkylcellulose, hydroxyalkylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, carboxymethylcellulose, and their cationic salts. Other examples of carriers include synthetic and natural bioceramics such as, but not limited to calcium carbonates, calcium phosphates, apatites, bioactive glass materials, and coral-derived apatites. See for example U.S. Patent Application 2002187104; PCT Int. Appl. WO/9731661; and PCT Int. Appl. WO/0071083, herein incorporated by reference.

In one embodiment, the carrier can further be coated by compositions, including bioglass and or apatites derived from sol-gel techniques, or from immersion techniques such as, but not limited to simulated body fluids with calcium and phosphate concentrations ranging from about 1.5 to 7-fold the natural serum concentration and adjusted by various means to solutions with pH range of about 2.8-7.8 at temperature from about 15-65 degrees C. See, for example, U.S. Pat. Nos. 6,426,114 and 6,013,591; and PCT Int. Appl. WO/9117965 herein incorporated by reference.

Other examples of carriers include, collagen (e.g. Collastat, Helistat collagen sponges), hyaluronan, fibrin, chitosan, alginate, and gelatin. See for example, PCT Int. Appls. WO/9505846; WO/02085422, herein incorporated by reference.

In one embodiment, the carrier can include heparin-binding agents; including but not limited to heparin-like polymers e.g. dextran sulfate, chondroitin sulfate, heparin sulfate, fucan, alginate, or their derivatives; and peptide fragments with amino acid modifications to increase heparin affinity. See for example, Journal of Biological Chemistry (2003), 278(44), p. 43229-43235, herein incorporated by reference.

In one embodiment, the composition can be in the form of a liquid, solid or gel. In one embodiment, the substrate can include a carrier that is in the form of a flowable gel. The gel can be selected so as to be injectable, such as via a syringe at the site where cartilage formation is desired. The gel can be a chemical gel which can be a chemical gel formed by primary bonds, and controlled by pH, ionic groups, and/or solvent concentration. The gel can also be a physical gel which can be formed by secondary bonds and controlled by temperature and viscosity. Examples of gels include, but are not limited to, pluronics, gelatin, hyaluronan, collagen, polylactide-polyethylene glycol solutions and conjugates, chitosan, chitosan & b-glycerophosphate (BST-gel), alginates, agarose, hydroxypropyl cellulose, methyl cellulose, polyethylene oxide, polylactides/glycolides in N-methyl-2-pyrrolidone. See for example, Anatomical Record (2001), 263(4), 342-349, herein incorporated by reference.

In one embodiment, the carrier can be photopolymerizable, such as by electromagnetic radiation with wavelength of at least about 250 nm. Example of photopolymerizable polymers include polyethylene (PEG) acrylate derivatives, PEG methacrylate derivatives, propylene fumarate-co-ethylene glycol, polyvinyl alcohol derivatives, PEG-co-poly(-hydroxy acid) diacrylate macromers, and modified polysaccharides such as hyaluronic acid derivatives and dextran methacrylate. See for example, U.S. Pat. No. 5,410,016, herein incorporated by reference.

In one embodiment, the composition can include a carrier that is temperature sensitive. Examples include carriers made from N-isopropylacrylamide (NiPAM), or modified NiPAM with lowered lower critical solution temperature (LCST) and enhanced peptide (e.g. NELL-1) binding by incorporation of ethyl methacrylate and N-acryloxysuccinimide; or alkyl methacrylates such as butylmethacrylate, hexylmethacrylate and dodecylmethacrylate. PCT Int. Appl. WO/2001070288; U.S. Pat. No. 5,124,151 herein incorporated by reference.

In one embodiment, where the carrier can have a surface that is decorated and/or immobilized with cell adhesion molecules, adhesion peptides, and adhesion peptide analogs which can promote cell-matrix attachment via receptor mediated mechanisms, and/or molecular moieties which can promote adhesion via non-receptor mediated mechanisms binding such as, but not limited to polycationic polyamino-acid-peptides (e.g. poly-lysine), polyanionic polyamino-acid-peptides, Mefp-class adhesive molecules and other DOPA-rich peptides (e.g. poly-lysine-DOPA), polysaccharides, and proteoglycans. See for example, PCT Int. Appl. WO/2004005421; WO/2003008376; WO/9734016, herein incorporated by reference.

In one embodiment, the carrier can include various naturally occurring matrices or their components such as devitalized cardiac matrix, vascular matrix, or other components derived from allograft, xenograft, or any other naturally occurring material derived from Monera, Protista, Fungi, Plantae, or Animalia kingdoms.

In one embodiment, the carrier can include one or more sequestering agents such as, but not limited to, collagen, gelatin, hyaluronic acid, alginate, poly(ethylene glycol), alkylcellulose (including hydroxyalkylcellulose), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, blood, fibrin, polyoxyethylene oxide, calcium sulfate hemihydrate, apatites, carboxyvinyl polymer, and poly(vinyl alcohol). See for example, U.S. Pat. No. 6,620,406, herein incorporated by reference.

In one embodiment, the carrier can include surfactants to promote stability and/or distribution of the NELL-1 peptide within the carrier materials such as, but not limited to polyoxyester (e.g. polysorbate 80, polysorbate 20 or Pluronic F-68).

In one embodiment, the carrier can include buffering agents such as, but not limited to glycine, glutamic acid hydrochloride, sodium chloride, guanidine, heparin, glutamic acid hydrochloride, acetic acid, succinic acid, polysorbate, dextran sulfate, sucrose, and amino acids. See for example, U.S. Pat. No. 5,385,887, herein incorporated by reference. In one embodiment, the carrier can include a combination of materials such as those listed above. By way of example, the carrier can a be PLGA/collagen carrier membrane. The membrane can be soaked in a solution including NELL-1 peptide.

An implant can include a substrate formed into the shape of a stent, mesh, pin, screw, plate, or prosthetic joint. An implant can include a substrate that is resorbable, such as a substrate including collagen.

The composition can also include an acceptable carrier to form a pharmacological composition. Acceptable carriers can contain a physiologically acceptable compound that acts, for example, to stabilize the composition or to increase or decrease the absorption of the agent. Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the anti-mitotic agents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would appreciate that the choice of a carrier, including a physiologically acceptable compound depends, for example, on the route of administration.

The compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable can include powder, or injectable or moldable pastes or suspension.

The compositions of this invention can comprise a pharmaceutically acceptable carrier, such as an aqueous carrier for water-soluble peptides. A variety of carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.

The concentration of NELL-1 peptide in these formulations can vary widely, and are selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.

The composition can include the PSC or iPS in various density of population. The PSC or iPS can have a density from about 1×10⁴ to about 1×10⁸/mL (per 1 mL volume of the composition). In some embodiments, the seeding density can be from about 1×10⁴ to about 1×10⁶, from about 1×10⁴ to about 1×10⁵, from about 1×10⁵ to about 1×10⁷, from about 1×10⁵ to about 1×10⁶, from about 1×10⁶to about 1×10⁷/ml, or from about 1×10⁷to about 1×10⁸/ml. Examples of seeding densities can be, e.g., 0.5×10⁴, 1×10⁴, 0.5×10⁵, 1×10⁵, 0.5×10⁶, 1×10⁶, 0.5×10⁷, 1×10⁷, or 1×10⁸/ml.

The dosage regimen for NELL-1 are determined by the clinical indication being addressed, as well as by various patient variables (e.g., weight, age, sex) and clinical presentation (e.g. extent of injury, site of injury, etc.). Generally, the NELL-1 is in a concentration sufficient to cause a PSC or iPS to differentiate into cardiac tissue (e.g., cardiac muscle cell or vascular cell) or progenitor cells so as to achieve cardio tissue regeneration (e.g., cardiomyo regeneration or cardiovascular regeneration).

Dosages of NELL-1 can be determined according to methods known in the art based on type of agent, the disease, and other factors such as age and gender.

In one embodiment, the dosage of NELL-1 factor can be described in terms of an amount per unit area of a composition or per unit weight of a composition. The dosage of NELL-1 generally ranges from 0.001 pg/mm² to 1 pg/mm², or more preferably from 0.001 ng/mm² to 1 ng/mm², or more preferably from 0.001 μg/mm² to 1 μg/mm², or more preferably from 0.001 mg/mm² to 1 mg/mm², or more preferably from 0.001 g/mm² to 1 g/mm², with or without a particular carrier or scaffold. In another embodiment, the dosage of NELL-1 generally ranges from 0.001 pg/ml to 1 pg/ml, or more preferably from 0.001 ng/ml to 1 ng/ml, or more preferably from 0.001 μg/ml to 1 μg/ml, or more preferably from 0.001 mg/ml to 1 mg/ml, or more preferably from 0.001 g/ml to 100 g/ml, with or without a particular carrier or scaffold. In yet another embodiment, the dosage of NELL-1 generally ranges from 0.001 pg/kg to 1 pg/kg, or more preferably from 0.001 ng/kg to 1 ng/kg, or more preferably from 0.001 μg/kg to 1 μg/kg, or more preferably from 0.001 mg/kg to 1 mg/kg, or more preferably from 0.001 gm/kg to 1 gm/kg, more preferably from 0.001 kg/kg to 1 kg/kg with or without a particular carrier or scaffold.

In some embodiments, the NELL-1 dosage can be described in terms of an amount per kilogram of body weight. For example, for the administration to adult humans, the dosage of NELL-1 can range from about 0.1 μg to about 100 mg per kilogram of body weight. In some embodiments, a suitable dose of a NELL-1 composition for the administration to adult humans is in the range of about 0.001 mg to about 20 mg per kilogram of body weight. In some embodiments, a suitable dose of a NELL-1 composition for the administration to adult humans is in the range of about 0.005 mg to about 10 mg per kilogram of body weight. In some embodiments, a suitable dose of a NELL-1 composition for the administration to adult humans is in the range of about 0.01 mg to about 5 mg per kilogram of body weight. In some embodiments, a suitable dose of a NELL-1 composition for the administration to adult humans is in the range of about 0.05 mg to about 1.0 mg per kilogram of body weight. However, the precise dosage to be therapeutically effective and non-detrimental can be determined by those skilled in the art.

Furthermore, it is understood that all dosages can be continuously given or divided into dosages given per a given timeframe. Examples of timeframes include but are not limited to every 1 hour, 2 hour, 4 hour, 6 hour, 8 hour, 12 hour, 24 hour, 48 hour, or 72 hour, or every week, 2 weeks, 4 weeks, or every month, 2 months, 4 months, and so forth.

Implantable Devices

Implantable devices shall encompass any implants for cardiac regeneration. An example of implantable device is stent. Stents are used not only as a mechanical intervention in vascular conditions, but also as a vehicle for providing biological therapy. As a mechanical intervention, stents act as scaffoldings, functioning to physically hold open and, if desired, to expand the wall of the passageway. Typically, stents are capable of being compressed, so that they can be inserted through small vessels via catheters, and then expanded to a larger diameter once they are at the desired location. Examples in patent literature disclosing stents that have been applied in PTCA (Percutaneous Transluminal Coronary Angioplasty) procedures include stents illustrated in U.S. Pat. No. 4,733,665 issued to Palmaz, U.S. Pat. No. 4,800,882 issued to Gianturco, and U.S. Pat. No. 4,886,062 issued to Wiktor.

U.S. patent application No. 20080188926 discloses a stem cell coated stent for cardiovascular conditions. The teaching in this application is incorporated herein by reference in its entirety.

Stents can be of virtually any design. Generally, the stent can include a supporting body, which provides a means to open up and support a lumen such as a blood vessel. Stents can be made of a metallic material or an alloy such as, but not limited to, cobalt chromium alloy (ELGILOY), stainless steel (316L), high nitrogen stainless steel, e.g., BIODUR 108, cobalt chrome alloy L-605, “MP35N,” “MP20N,” ELASTINITE (Nitinol), tantalum, nickel-titanium alloy, platinum-iridium alloy, gold, magnesium, or combinations thereof “MP35N” and “MP20N” are trade names for alloys of cobalt, nickel, chromium and molybdenum available from Standard Press Steel Co., Jenkintown, Pa. “MP35N” consists of 35% cobalt, 35% nickel, 20% chromium, and 10% molybdenum. “MP20N” consists of 50% cobalt, 20% nickel, 20% chromium, and 10% molybdenum. Devices made from bioabsorbable or biostable polymers could also be used with the embodiments of the present invention.

Stents can also include a coating. The coating can include a matrix layer, which can include NELL-1 protein and optionally an anti-inflammatory agent and/or an anti-proliferative agent. The matrix layer can be formed from a biodegradable or biodurable material. Examples of such biodegradable materials include natural polymers (e.g., polysaccharides or protein polymer such as collagen) or synthetic biodegrable polymers.

Any biocompatible polymers can be used to form a coating on a stent. Such biocompatible polymers include, but not limited to, poly(ester amide), poly(ester amide) that may contain alkyl groups, amino acid groups, or poly(ethylene glycol) (PEG) groups, polyethylene glycol (PEG), polylakanoates (PHA), poly(2-hydroxyalkanoates), poly(3-hydroxyalkanoates) such as poly(3-hydroxypropanoate), poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), poly(3-hydroxyhexanoate), poly(3-hydroxyheptanoate) and poly(3-hydroxyoctanoate), poly(4-hydroxyalknaote) such as poly(4-hydroxybutyrate), poly(4-hydroxyvalerate), poly(4-hydroxyhexanote), poly(4-hydroxyheptanoate), poly(4-hydroxyoctanoate) and copolymers comprising any of the 3-hydroxyalkanoate or 4-hydroxyalkanoate monomers described herein or blends thereof, polyesters, poly(D,L-lactide), poly(L-lactide), polyglycolide, poly(D,L-lactide-co-glycolide), polycaprolactone, poly(D,L-lactide-co-caprolactone), poly(glycolide-co-caprolactone), poly(dioxanone), poly(ortho esters), poly(anhydrides), poly(tyrosine carbonates) and derivatives thereof, poly(tyrosine ester) and derivatives thereof, poly(imino carbonates), poly(phosphoesters), polyphosphazenes, poly(amino acids), polysaccharides, collagen, chitosan, alginate, polyethers, polyamides, polyurethanes, polyalkylenes, polyalkylene oxides, polyethylene oxide, polypropylene oxide, polyethylene glycol (PEG), PHA-PEG, polyvinylpyrrolidone (PVP), alkylene vinyl acetate copolymers such as ethylene vinyl acetate (EVA), alkylene vinyl alcohol copolymers such as ethylene vinyl alcohol (EVOH or EVAL), poly(n-butyl methacrylate) (PBMA), SOLEF™ polymers such as poly(vinylidene fluoride-co-hexafluoropropene) (PVDF-co-HFP) and poly(vinylidene fluoride) (PVDF) and combinations thereof. In some embodiments, the polymer can be acrylate or methacrylate based polymers or copolymers. In some embodiments, the polymers can include phosphoryl choline pendant groups or moieties.

In some embodiments, the support body or the coating can include a plurality of pores. The pores can be used to load PSC or iPS. The PSC or iPS can be loaded onto a stent prior to use of the stent by, e.g., immersing a stent, with or without holes, to a PSC or iPS cell suspension.

Method of Fabrication

Generally, the method of making the composition comprises the acts of

a) providing a cardioinductive agent (e.g., NELL-1 protein),

b) providing a population of PSC or iPS, and

c) forming the composition,

wherein the cardioinductive agent is in (1) a therapeutically effective amount for causing PSC or iPS to differentiate in the cardiac cell or progenitor lineages so as to generate cardio tissues or (2) a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth.

In some embodiments, forming comprises forming a formulation comprising the NELL-1 protein, and seeding the formulation with a population of the PSC or iPS. In some embodiments, the formulation can further comprise an excipient, e.g., which is further described below.

The formulation can take any dosage form. In some embodiments, its is a powder formulation. In some embodiments, it is a liquid formulation. In some embodiments, it is a semi-solid/semi-liquid formulation, e.g., a gel or paste. In some embodiments, the formulation is an implantable device such as a bone implant. In some embodiments, the formulation is a scaffold.

The formulation can take any desirable form for seeding a population of PSC or iPS. In some embodiments, where the formulation is a scaffold or an implant, the formulation can be porous for seeding the PSC or iPS. The pores in the formulation can have a volume that is capable of accommodating the seeding density of the PSC or iPS.

Seeding of the PSC or IPS can be achieved by well established cell seeding procedures (see, e.g., Undale, et al., Mayo Clin Proc., 84(1):893-902 (2009); Cancedda et al., Biomaterials 28: 4240-4250 (2007); and Marcacci, et al., Tissue Engineering, 13(5):947-955) (2007)) and can vary according to the dosage form of the composition. For example, for liquid formulations, seeding can be readily achieved by placing a population in the formulation. An example of seeding a porous implant or scaffold is documented (data not shown).

The seeding density for PSC or iPS can vary from about 1×10⁴to about 1×10⁸/mL (per 1 mL volume of the composition). In some embodiments, the seeding density can be from about 1×10⁴to about 1×10⁶, from about 1×10⁴to about 1×10⁵, from about 1×10⁵to about 1×10⁷, from about 1×10⁵ to about 1×10⁶, from about 1×10⁶ to about 1×10⁷/ml, or from about 1×10⁷to about 1×10⁸/ml. Examples of seeding densities can be, e.g., 0.5×10⁴, 1×10⁴, 0.5×10⁵, 1×10⁵, 0.5×10⁶, 1×10⁶, 0.5×10⁷, 1×10⁷, or 1×10⁸/ml.

The following describes an example of seeding the PSC or iPS in a scaffold:

Seeding of pericytes. A hPSC suspension is seeded into the ES-TIPS PEUU scaffolds via a vacuum rotational seeding device described by Soletti et al., Biomaterials, 27(28):4863-70 (2006). In brief, a scaffold is connected to two coaxial stainless steel tees inside an airtight chamber. The chamber is connected to house vacuum to maintain a negative and constant pressure (−130 mmHg), which induces transmural flow of the cell suspension across the scaffold. A syringe pump infuses the cell suspension (1×10⁶ cells/mL) at 2.5 mL/min into the manually rotating scaffold to reach 3^(x)10⁶ cells per scaffold within less than 2 min, resulting in a scaffold with uniformly distributed cells. Seeded scaffolds are immediately put into static culture for 3 hours, which is sufficient for cellular attachment (see, e.g., Soletti, et al., Biomaterials, 29(7):825-33 (2008)). Scaffolds are then transferred to a spinner flask with 200 mL medium at 15 rpm stirring for 2 days of culture, after which it was implanted into the rat.

Scaffolds are observed under SEM immediately after seeding. The seeded scaffolds after 2 days' dynamic culture are further assessed for histology (H&E), cellular distribution (F-actin and nuclear staining), and attachment (SEM). For F-actin staining, cryosections are permeabilized (0.1% Triton) for 30 min, blocked (2% BSA) for 30 min and then incubated with Alexa 488-conjugated phalloidin (1:500, Sigma) for 1 hour.

Methods of Use

Generally, a method of use of invention comprises administering to a mammal (e.g., a human subject) the composition of the various embodiments disclosed herein to treat, delay, or ameliorate a cardiac condition. The compositions may be administered at specific sites in or on tissues or organs. The compositions are administered in an amount effective to treat the specified condition or disorder, which are also referred to as cardiac conditions or damages, which can be acute or chronic. Common cardiac conditions include, e.g., congenital heart disease, aortic aneurysms, aortic dissections, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, heart attack, or myocardial infarction.

The compositions may be administered by various methods know to the skilled artisan. In some embodiments, compositions are administered by injection, such as with a hypodermic needle. The size (Le., gauge) of the hypodermic needle will depend on factors such as the type of composition, the amount being injected, the spatial location for depositing the composition. Typical gauges for injection are available from 12 to 25 gauge of various lengths.

In other embodiments, the compositions are administered using a catheter. The catheter may be a flexible, rigid, or semi rigid tube or conduit positioned at the site for deposition of the cultured tissues. The catheter may be made of various materials, non-limiting example of which include, among others, plastic, metal, and silicon. Where the compositions comprise cords or sutures, they made be administered by injection or catheter, but may also be introduced into tissue sites by methods typically used for suturing tissues, e.g., using an attached suturing needle. The cord or suture is threaded into the tissue and then left in place by detaching the suturing needle.

In still other embodiments, an incision is made in the tissue or organ, and the compositions applied into the incision site. The compositions may be held in place by suturing the tissue or organ at the site of the incision to cover and contain the compositions. Placement of the compositions may be done during surgery to repair damaged tissue, which may enhance repair of the surgical damage as well as the tissue damaged by a disorder or disease.

In some embodiments, the administration of the compositions may be guided by various medical imaging techniques, including, but not limited to, ultrasound, fiber optic, magnetic resonance imaging, or computer assisted tomography. As noted above, the three dimensional framework may have contrast agents to assist in imaging of the compositions as it is administered into the subject.

The dosages for administration will take into consideration various factors such as the nature of the condition being treated, the type of tissue or organ, the amount that the tissue or organ can accommodate, degradation properties of the three dimensional scaffold in vivo, duration of cell activity following administration, and the level of growth factors produced. Three dimensional frameworks that degrade at a faster rate may be administered at a higher frequency without significant accumulation of the framework material in the tissue or organ while materials with slower degradation rates may be administered with lower frequency to limit the amount of undegraded material present in the injected site. The frequency of administration may also be adjusted for elimination of the framework material by bodily mechanisms, such as through systemic circulation and the lymphatic system.

In various embodiments, the compositions may be administered once per day, about twice per week, about once per week, about once every two weeks, about once every month, or about once per six months, or more or less depending, at least in part, on the factors discussed above. The compositions may be administered at different sites concurrently or sequentially. When administered at different sites, the administrations may be to a localized area. The spatial density of administration in a localized area may depend on the extent of the tissue or organ being treated, such as volume and surface area as well as depth of the treated site.

When administering within a tissue or organ, injection may be done at the same depth or at different depths. In some embodiments, the compositions are administered into a body cavity, either naturally occurring or induced by injury, disease, surgery, or other conditions described above.

In some embodiments, the compositions are used to treat acute tissue damage. As used herein, “acute damage” refers to damage or wounds caused by, among others, traumatic force, chemical toxicity, thermal burns, frostbite, acute ischemia, and reperfusion injury. Exemplary traumatic force injury includes, among others, surgical procedures and blunt force trauma (e.g., gun shot wounds, knife wounds, etc.). The compositions may be applied on or injected into the affected tissues to promote vascularization, repair, and regeneration of such damaged tissues.

In other embodiments, the compositions are used to treat chronic tissue damage. As used herein, “chronic tissue damage” refers to tissue damage resulting from persistent or repeated insults to a tissue, typically showing manifestations of persistent or chronic inflammatory reaction or unhealed or improper healing of tissue. Chronic tissue damage may also be characterized by the presence tissue remodeling, such as fibrosis, known as scarring, originating from the repeated insult. Other forms of tissue remodeling in chronic tissue damage include, among others, thickening of tissue arising from compensatory changes to reduced tissue function, or tissue thinning where cytopathic effects result in continual loss of cells without compensatory cell renewal. Some chronic tissue damage may show tissue thinning during the early stages of damage followed by tissue thickening arising from repeated scarring and/or compensation for reduced tissue function. Chronic tissue damage may arise in many different contexts, such as repeated exposure to irritants or toxic chemicals, persistent or repeated ischemic events (e.g., chronic ischemia, micro-strokes), chronic infections, and persistent disease condition (e.g., autoimmune disease, ulcers, atherosclerosis, congenital defects, etc.).

In some embodiments, the tissue damage comprises ischemic damage. As used herein, “ischemic tissue” refers to tissues that have been deprived of blood or oxygen supply, thereby resulting in injury to cells and tissues. On the cellular level, ischemia is any process in which there is a lack of sufficient blood flow to a portion of the tissue, thereby initiating an ischemic cascade, leading to the death of cells. For instance, myocardial ischemia is a condition in which oxygen deprivation to the heart muscle is accompanied by inadequate removal of metabolites because of reduced blood flow or perfusion. Myocardial ischemia can occur as a result of increased myocardial oxygen demand, reduced myocardial oxygen supply, or both. Myocardial ischemia may be caused by reduction of oxygen supply secondary to increased coronary vascular tone (i.e., coronary vasospasm) or by marked reduction or cessation of coronary flow as a result of platelet aggregates or thrombi.

“Acute ischemia” refers to an abrupt or sudden disruption in blood flow to tissues. For instance, acute ischemia in the heart, also known as myocardial infarction, is generally caused by a rapid occlusion of the coronary arteries, such as that arising from ruptured proximal arteriosclerotic plaque, acute thrombosis on preexisting atherosclerotic disease, an embolism from the heart, aorta, or other large blood vessel, or a dissected aneurysm. In embodiments in which acute ischemia is diagnosed, the compositions may be applied onto or into the area of ischemically damaged tissue to promote vascularization, increase blood flow to the muscles and promote regeneration of heart tissue.

“Chronic ischemia” typically refers to disruption in blood flow to tissues by gradual enlargement of an atheromatous plaque that reduces blood flow to the affected downstream tissue. As cells die and the tissue becomes damaged, remodeling may occur, such as tissue thinning from cell death, and tissue thickening and disorganization from scarring events arising from cellular response to the damage.

In some embodiments, the compositions are used to treat an ischemically damaged heart tissue, various forms of which include, among others, acute myocardial ischemia, chronic myocardial ischemia, and congestive heart failure. Other disorders of the heart, such as cardiomyopathy, may also be treated with the compositions described herein. As noted above, cardiovascular ischemia may be caused by a rupture of an atherosclerotic plaque in a coronary artery, leading to formation of thrombus, which can occlude or obstruct a coronary artery, thereby depriving the downstream heart muscle of oxygen. Necrosis resulting from the ischemia is commonly called an infarct.

Chronic ischemia in the heart is believed to occur by gradual enlargement of an atheromatous plaque that reduces blood flow to the heart. As the heart weakens, remodeling occurs, typically in the ventricles, and the heart enlarges and becomes rounder. The heart also undergoes changes at the cell level characterized by cell apoptosis, resulting in a less distensible heart and a weakening of the heart muscle over time.

“Congestive heart failure” refers to impaired cardiac function in which the heart fails to maintain adequate circulation of blood, and in some embodiments, is the end result of damage from chronic ischemia. The most severe form of congestive heart failure leads to pulmonary edema, which develops when this impairment causes an increase in lung fluid secondary to leakage from pulmonary capillaries into the interstitium and alveoli of the lung. In some embodiments, heart function in congestive heart failure is expressed as an imbalance in the degree of end-diastolic fiber stretch proportional to the systolic mechanical work expended in an ensuing contraction (also known as the Frank-Starling principle). Various parts of the heart may be affected, including left ventricle and right ventricle.

“Cardiac myopathy” is typically defined by any structural or functional abnormality of the ventricular myocardium, except for congenital developmental defects, valvular disease; systemic or pulmonary vascular disease; isolated pericardial, nodal, or conduction system disease; or epicardial coronary artery disease; unless chronic diffuse myocardial dysfunction is present. Based upon clinical indications, the disorder may be diagnosed as dilated congestive, hypertrophic, or restrictive cardiomyopathy. Dilated congestive cardiomyopath is generally characterized chronic myocardial fibrosis with diffuse loss of myocytes. Without being limited by theory, the underlying pathologic process is believed to start with an acute myocarditic phase, which may have viral causes, followed by a variable latent phase, then a phase of chronic fibrosis and death of myocardial myocytes due to an autoimmune reaction to virus-altered myocytes. Whatever the cause of the disorder, it leads to dilation, thinning, and compensatory hypertrophy of the remaining myocardium interspersed with fibrosis. Functionally, there is impaired ventricular systolic function reflected by a low ejection fraction (EF). Hypertrophic cardiomyopathy is characterized by marked ventricular hypertrophy with diastolic dysfunction. At the cellular level, the cardiac muscle is abnormal with cellular and myofibrillar disarray. The most common asymmetric form of hypertrophic cardiomyopathy displays marked hypertrophy and thickening of the upper interventricular septum below the aortic valve. The hypertrophy results in a stiff, noncompliant chamber that resists diastolic filling, leading to elevated end-diastolic pressure, which raises pulmonary venous pressure. Restrictive cardiomyepathy is characterized by rigid, noncompliant ventricular walls that resist diastolic filling of one or both ventricles, most commonly the left. This less frequent form of cardiacmyopathy has different causes, often being associated with other disorders or conditions, such as Gaucher's Disease, Loffler's Disease, amyloidosis, and endorcardial fibrosis. Physiology of the heart shows endocardial thickening or myocardial infiltration with loss of myocytes, compensatory hypertrophy, and fibrosis, all of which may lead to atrioventricular valve malfunction. Functionally, the heart shows diastolic dysfunction with a rigid, noncompliant chamber with a high filling pressure. Systolic function may deteriorate if compensatory hypertrophy is inadequate in cases of infiltrated or fibrosed chambers.

The mode of administration can be implanting, direct injection, coating on metallic or non-metallic artificial implants, or placing around an implant (e.g., a metallic or non-metallic artificial implant). Some examples of delivering the composition can be, e.g., percutaneous injection through intact skin to various sites, or direct injection through nonintact skin (e.g., surgically opened sites or trauma sites). In some embodiments, the delivery can be surgical implantation of a composition described herein. In some embodiments, the delivery can be one of extravascular delivery, injection or catheter based injections; intravascular delivery, injection or catheter based injections; intravenous delivery, injection or catheter based injections; intraarterial delivery, injection or catheter based injections; intrathecal delivery, injection or catheter based injections; intraosseous delivery, injection or catheter based injections; intracartilaginous delivery, injection or catheter based injections; or intravesical delivery, injection or catheter based injections.

In some embodiments, a delivery of composition described herein to a mammalian subject can be delivery via mechanical pumps with percutaneous or implantable catheters.

In some embodiments, a delivery of composition described herein to a mammalian subject can be catheter based delivery to any area/organ in the body.

In some embodiments, a delivery of composition described herein to a mammalian subject can be delivery via expanded dispersion through various devices promoting increased tissue penetration or wider tissue distribution (e.g., ultrasound, iontophoresis, heat, pressure, etc.).

EXAMPLES

The embodiments of the present invention are illustrated by the following set forth examples. All parameters and data do not limit the scope of the embodiments of the invention.

Example 1 Isolation of Adventitia PSC from Liposuction Aspirate

SVF Isolation from Liposuction Aspirate.

Liposuction aspirate was washed with an equal volume of PBS and centrifuged for 10 minutes at 400×g. After centrifugation, the top layer of the preparation, representing the tissue fraction containing the SVF, was collected and further washed in PBS. The tissue fraction was then enzymatically processed by addition of an equal volume of digestion solution (DMEM, collagenase II 1 mg/ml, DNAse 10 μg/ml, 1% Pen-Strepto, 3% BSA) and incubation for 45 minutes at 37 C under agitation (250 rpm). The enzymatic digestion was stopped after addition of PBS 5mM EDTA and the solution was filtered through 100 μm cell strainer. After two washes in PBS 5mM EDTA and centrifugation at 400×g for 10 minutes, the supernatant containing adipocytes was discarded and the pellet was resuspended in 10 ml red blood cell lysis buffer for 10 minutes at room temperature. The suspension was washed in PBS 5mM EDTA and centrifuged at 400×g for 10 min. The pellet containing the SVF was resuspended in DMEM 10% FBS prior to cell count, culture and staining with specific antibodies for the purification of PSC via FACS sorting.

SVF Culture and CFU-F Assay

Conventional MSC-like adipose stem cells (ASC) was isolated by plating 10⁷ unfractionated SVF cells per well in a 6 multiwell plate. CFU-F assay was performed for the determination of the frequency of ASC by limiting dilution of total unfractionated SVF cell plated at the increasing density of 10² to 10⁶ cells per well in a 6 multiwell plate. After 2 weeks of culture, plates were stained with May Grumwald/Giemsa and the number of colonies was scored.

Detection of PSC within the Liposuction-derived SVF

Three hundred thousand SVF cells were washed and re-suspended in 100 μl of PBS before incubation at 4 C for 20 minutes with DAPI for the exclusion of dead cells and with the following antibodies: CD45,CD34,CD31,CD146. Cells were then washed and resuspended in PBS prior to flow cytometry analysis. After exclusion of dead cells and CD45+hematopoietic cells, PSC are identified as CD146+CD34− pericytes and CD34+CD31−CD146− adventitial cells. Endothelial cells are instead defined as CD34+CD146+CD31+cells. The same staining procedure was used for the isolation of PSC via FACS sorting. Purified PSC were resuspended in EGM2 medium and plated in 0.2% gelatin coated wells at the density of 2×10⁴ cells per cm². CFU-F assay were performed as above described.

Example 2 Cardiogenic Studies on Pericytes and NELL-1

Studies on cardiogenic ability of pericytes and NELL-1 factor were performed in a SCID mouse thigh muscle implantation model. The results are shown in FIGS. 1 and 2. FIG. 1 shows increased pericyte proliferation/survival when Nell-1 is added. FIG. 2 shows increased VEGF expression by pericytes when Nell-1 is added.

The above results clearly documented the cardiogenic ability of pericytes and NELL-1. These experiments demonstrate the effects of NELL-1 for enhancing survivability and engraftment of PSC or iPS and for causing PSC or iPS to differentiate into cardiac cell or progenitor cell lineage so as to generate a cardio tissue.

While particular embodiments of the present invention have been shown and described, it are obvious to those skilled in the art that changes and modifications can be made without departing from this invention in its broader aspects. Therefore, the appended claims are to encompass within their scope all such changes and modifications as fall within the true spirit and scope of this invention

REFERENCES

-   1. WRITING GROUP MEMBERS, Lloyd-Jones D, Adams R J, Brown T M,     Carnethon M, Dai S, et al. Heart Disease and Stroke Statistics—2010     Update: A Report From the American Heart Association. Circulation     2010;121(7):e46-215. -   2. Soletti L, Hong Y, Guan J, Stankus J J, El-Kurdi M S, Wagner W R,     et al. A bilayered elastomeric scaffold for tissue engineering of     small diameter vascular grafts. Acta Biomater 2010;6(1):110-22. -   3. Matsuda T. Recent progress of vascular graft engineering in     Japan. Artif Organs 2004;28(1):64-71. -   4. Niklason LES, M (2002) Small-Diameter Vascular Grafts. Methods of     Tissue Engineering, ed Atala A L, R. P (Academic Press, San Diego),     p 905. -   5. Seal B L, Otero T C, Panitch A. Polymeric biomaterials for tissue     and organ regeneration. Mat Sci Eng R 2001;34(4-5):147-230. -   6. Kakisis J D, Liapis C D, Breuer C, Sumpio B E. Artificial blood     vessel: the Holy Grail of peripheral vascular surgery. J Vasc Surg     2005;41(2):349-54. -   7. Thomas A C, Campbell G R, Campbell J H. Advances in vascular     tissue engineering. Cardiovasc Pathol 2003;12(5):271-6. -   8. Xue L, Greisler H P. Biomaterials in the development and future     of vascular grafts. J Vasc Surg 2003;37(2):472-80. -   9. Caplan A I. Adult mesenchymal stem cells for tissue engineering     versus regenerative medicine. J Cell Physiol 2007;213(2):341-7. -   10. Eberli D, Atala A (2006) Tissue engineering using adult stem     cells. Stem Cell Tools and Other Experimental Protocols, Methods in     Enzymology, (Elsevier Academic Press Inc, San Diego), Vol 420, pp     287-302. 

1. A composition, comprising: a population of perivascular stem cells (PSCs) or induced pluripotent stem cells, and a cardioinductive agent, wherein the cardioinductive agent is in: a therapeutically effective amount for causing PSC or iPS to differentiate in the cardiac cell or progenitor lineages so as to generate cardio tissues; or a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth.
 2. The composition of claim 1, wherein the agent is a NELL-1 protein.
 3. The composition of claim 1, wherein the PSC or iPS has a density of about 1×10⁴ to about 1×10⁸/per 1 mL volume of the composition.
 4. The composition of claim 1, wherein the PSC is pericytes or adventitia cells.
 5. The composition of claim 2, which is a scaffold.
 6. The composition of claim 5, wherein the scaffold comprises a porous body, wherein the NELL-1 protein is embedded in the body of scaffold, and wherein the PSC or iPS is seeded in the pores in the scaffold.
 7. The composition of claim 6, which is a vascular graft.
 8. The composition of claim 5, wherein the scaffold is biodegradable or biodurable.
 9. The composition of claim 6, wherein the scaffold is biodegradable or biodurable.
 10. An implantable device, comprising: a population of perivascular stem cells (PSCs) or induced pluripotent stem cells, and a cardioinductive agent, wherein the cardioinductive agent is in: a therapeutically effective amount for causing PSC or iPS to differentiate in the cardiac cell or progenitor lineages so as to generate cardio tissues; or a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth.
 11. The implantable device of claim 10, wherein the agent is a NELL-1 protein.
 12. The implantable device of claim 10, wherein the PSC or iPS has a density of about 1×10⁴to about 1×10⁸/per 1 mL volume of the device.
 13. The implantable device of claim 10, wherein the PSC is pericytes or adventitia cells.
 14. The implantable device of claim 11, which is a stent.
 15. The implantable device of claim 14, wherein the stent comprises a supporting body and an optional coating, and wherein the PSC or iPS and NELL-1 protein are included in the supporting body or the optional coating.
 16. The implantable device of claim 15, wherein the support body or the optional coating comprises a plurality of pores, wherein the NELL-1 protein is embedded in the support body or the optional coating, and wherein the PSC or iPS is seeded in the pores.
 17. The implantable device of claim 15, wherein the optional coating is biodegradable or biodurable.
 18. The implantable device of claim 16, which is biodegradable or biodurable.
 19. A method of fabricating a composition or implantable device, comprising: providing a population of perivascular stem cells (PSC) or induced pluripotent stem cells (iPS), providing a cardioinductive agent, and forming the composition or implantable device, wherein the cardioinductive agent is in: a therapeutically effective amount for causing PSC or iPS to differentiate in the cardiac cell or progenitor lineages so as to generate cardio tissues; or a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth.
 20. The method of claim 19, wherein the composition is according to claim
 1. 21. The method of claim 19, wherein the implant is according to claim
 10. 22. A method of treating or ameliorating a cardiac condition, comprising administering to a subject a composition in need thereof, the composition comprising: a population of perivascular stem cells (PSC) or induced pluripotent stem cells (iPS), a cardioinductive agent, wherein the cardioinductive agent is in: a therapeutically effective amount for causing PSC or iPS to differentiate in the cardiac cell or progenitor lineages so as to generate cardio tissues; or a therapeutically effective amount for enhancing the survivability or engraftment of the PSC or iPS where PSC or iPS provide trophic factors or enhance vascular ingrowth.
 23. The method of claim 22, wherein the subject is a human being, and wherein the cardiac condition is a cardiovascular condition or myocardial infarction (MI).
 24. The method of claim 22, wherein the composition is an implantable device. 